Abstract Pre-mRNA splicing, an essential step in human gene expression, is catalyzed by a large and dynamic RNA-protein complex called the spliceosome. An understanding of the structure and function of the spliceosome is critical to the development of therapeutics for splicing-associated diseases such as retinitis pigmentosa and myelodysplastic syndromes, and will provide insight into how RNAs and proteins cooperate to carry out cellular functions. U6 RNA is a core component of the spliceosome and undergoes dramatic rearrangements in conformation and binding partners during each splicing event. We aim to determine how the structure of U6 RNA confers its function throughout the splicing cycle, and will use the genetically tractable budding yeast Saccharomyces cerevisiae as our model system. Based on biochemical and genetic evidence, we hypothesize that the highly conserved U6 nucleotides A62 and C85 participate in important RNA-RNA and/or RNA-protein interactions in the splicing cycle that have not yet been characterized. To determine the function(s) of these residues, we will pursue three specific aims. In Aim 1, we will use biochemical assays to determine the arrest point of the mutations U6-A62U/C85A (U6-UA) and U6-A62C/C85G (U6-CG), and will conduct a genetic experiment to relate the mutants? defects to the function of a spliceosomal helicase. In Aim 2, we will look for mutations that suppress the defects of U6-UA and U6-CG using genome-wide selections and a selection targeted to the spliceosome?s ?master regulator? protein Prp8. In Aim 3, we will use mutagenesis, in vivo photocrosslinking, and pull-down assays to determine how mutations in Prp8 suppress the U6-UA defect. This experimentation will be guided by the wealth of structural information available in published models of spliceosomal complexes solved by cryo-electron microscopy. Our proposed research has the potential to (i) identify a novel cold-sensitive block in the splicing cycle, providing an experimental tool with which to better understand the mechanism of splicing, and (ii) reveal a cascade of molecular interactions important to ensure accurate splicing.